Initially known as cytokine synthesis inhibitor factor or CSIF, interleukin-10 (IL-10) is a potent immunomodulator of hematopoietic cells, particularly immune cells. Cells such as activated Th2 cells, B cells, keratinocytes, monocytes and macrophages produce IL-10. See, e.g., Moore et al., Annu. Rev. Immunol. 11:165 (1993). IL-10 inhibits activation and effector functions of a number of cells that include T cells, monocytes and macrophages. In particular, IL-10 inhibits cytokine synthesis, including that of IL-1, IFN-γ, and TNF, by cells such as Th1 cells, natural killer cells, monocytes, and macrophages. See, e.g., Fiorentino et al., J. Exp. Med., 170:2081-2095 (1989); Fiorentino et al., J. Immunol. 146:3444 (1991); Hsu et al., Int. Immunol. 4:563 (1992); Hsu et al., Int. Immunol. 4:563 (1992); D'Andrea et al., J. Exp. Med. 178:1041 (1993); de Waal Malefyt et al., J. Exp. Med. 174:915 (1991); Fiorentino et al., J. Immunol. 147:3815 (1991).
Multiple pathogens, particularly intracellular pathogens, elicit IL-10 production to slow or completely stall the effective removal of the pathogen by the immune response. Moore et al., Annu. Rev. Immunol. 11:165 (1993). For example, in blood lymphocytes from patients with HIV, leprosy, or tuberculosis, peripheral blood lymphocytes are typically anergic or nonresponsive in vitro when challenged with the pathogen. However, the neutralization of IL-10 in these demonstrated that an active effector response, i.e., Th1 reactivity, was present in these cells. Thus, it is believed that IL-10 is effectively commandeered by the pathogen to facilitate its infective state.
IL-10 is also associated with autoimmunity in vivo. Autoimmunity results from the development from autoantibodies, autoreactive T cells, or some combination thereof that target normal tissue. One example of autoimmune disease is systemic lupus erythematosus (SLE), a chronic rheumatic disease in which connective tissue throughout the body becomes inflamed. Autoantibodies that attack normal body tissue as if it were an outside invade result in the characteristic inflammation. While the precise cause is not fully understood, researchers believe it has both genetic and environmental components. Specifically, B-cell hyperactivity and the presence of various autoantibodies characterize SLE. Typically, IgG autoantibodies reactive to double stranded DNA (IgG anti-dsDNA abs) are elevated in patients with SLE. Between 60 and 70% of SLE patients produce IgG anti-dsDNA abs, some of which are nephrotoxic. SLE is ten times more prevalent in women than men, with symptoms ranging from facial rashes to disabling and potentially life-threatening organ dysfunction. It can develop at any age, but is most common in young adults.
Numerous studies support a role for IL-10 in the pathology associated with SLE. For example, while IL-10 is typically not produced by cells without appropriate stimulation, both B cells and macrophages from SLE patients spontaneously produce high levels of IL-10 in vitro. Llorente, et al., Arthritis Rheum. 40:249-60 (1997). In several studies, researchers demonstrated a correlation between serum levels of IL-10 and disease activity. Moreover, both in vivo and in vitro studies demonstrated that the blockade of IL-10 production can alleviate the clinical manifestations of SLE. See, e.g., Gonzalez-Amaro, et al. J. Autoimmunity 11:395-402 (1998).
To date, one of the manifestations of SLE, lupus nephritis, has been treated with through the use of immunosuppressive therapies, e.g., corticosteriods and cyclophosphamides. Although effective, these therapies are non-specific and substantial toxicities exist which prevent long term therapy. Thus, specific neutralizing antibodies may be effective antagonists of IL-10, permitting the removal of the suppressive effects of IL-10 while leaving the remainder of the immune response network intact.
The most significant limitation in using antibodies as a therapeutic agent in vivo is the immunogenicity of the antibodies. As most monoclonal antibodies are derived from rodents, repeated use in humans results in the generation of an immune response against the therapeutic antibody. Such an immune response results in a loss of therapeutic efficacy at a minimum and a potential fatal anaphylactic response at a maximum. Initial efforts to reduce the immunogenicity of rodent antibodies involved the production of chimeric antibodies, in which mouse variable regions were fused with human constant regions. Liu et al., Proc. Natl. Acad. Sci. USA 84:3439 (1987). However, mice injected with hybrids of human variable regions and mouse constant regions develop a strong anti-antibody response directed against the human variable region, suggesting that the retention of the entire rodent Fv region in such chimeric antibodies may still result in unwanted immunogenicity in patients.
It is generally believed that complementarity determining region (CDR) loops of variable domains comprise the binding site of antibody molecules. Therefore, the grafting of rodent CDR loops onto human frameworks (i.e., humanization) was attempted to further minimize rodent sequences. Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988). However, CDR loop exchanges still do not uniformly result in an antibody with the same binding properties as the antibody of origin. Changes in framework residues (FR), residues involved in CDR loop support, in humanized antibodies also are required to preserve antigen binding affinity. Kabat et al., J. Immunol. 147:1709 (1991). While the use of CDR grafting and framework residue preservation in a number of humanized antibody constructs has been reported, it is difficult to predict if a particular sequence will result in the antibody with the desired binding, and sometimes biological, properties. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA 86:10029 (1989), Gorman et al., Proc. Natl. Acad. Sci. USA 88:4181 (1991), and Hodgson, Bio/Technology 9:421 (1991). Moreover, most prior studies used different human sequences for animal light and heavy variable sequences, rendering the predictive nature of such studies questionable. Sequences of known antibodies have been used or, more typically, those of antibodies having known X-ray structures, antibodies NEW and KOL. See, e.g., Jones et al., supra; Verhoeyen et al., supra; and Gorman et al., supra. Exact sequence information has been reported for only a few humanized constructs.
The present invention provides humanized monoclonal antibodies which recognize human IL-10 and modulate its activity, in particular with regard to autoimmune disorders. The humanized antibody should provide an alternative therapy choice without the toxicity and non-specificity associated with current treatments.